The present invention relates to HIgR (herpesviris immunoglobulin-like receptor) for the manufacture of medicaments for preventing or treating HSV-1, HSV-2 infections.
The present invention also relates to a novel immunoglobulin-like protein and medicaments which incorporate said antibodies and are useful in preventing or treating pathologies concerned with herpes simplex virus 1, herpes simplex virus 2 infections in humans. The invention also relates to methods for obtaining cell lines resistant to HSV-1, HSV-2 and BHV-1 infections and to cell lines expressing specific receptors which mediate entry of said virus within cellular environment to test efficacy of antiherpes drugs and antibodies, antiherpesvirus vaccines and herpesvirus-based vectors.
Alphaherpesviruses which includes HSV-1, HSV-2, PRV, and BHV-1, infect a variety of cells resulting in efficient virus production in a short replicative cycle. Infection in the mammals results in lesion of the mucocutaneous tissue, and specifically in humans produces lesion on the mucosal surfaces, with spread of virus to the nervous system and establishment of latent infections in neurons. Infections may lead in some cases, to encephalitis or meningitis, with an often fatal outcome.
The receptors which mediate herpes simplex virus (HSV) entry into cells have remained elusive for a long time for several reasons.
It has been known that binding of alphaherpesviruses to cells occurs through an interaction of virion glycoproteins gC with cell surface glycosaminoglycans, whereas the following entry of the capsid into the cytoplasm occurs via a fusion of the virion envelope with cell membrane. This last step involves at least the four glycoproteins gB, gD, and the heterodimer gH, gL.
Works of the past have allowed to ascertain that alphaherpesvirus gD is the virion component that interacts with a cell surface receptor. One cellular receptor which mediates virus entry was called herpesvirus entry mediator (HVEM), and has been redesignated HveA.
The molecular mechanisms underlying this process remain in part obscure, and a model is still lacking of how the interaction of gD with its cellular receptor triggers the fusion of the virion envelope with the plasma membrane and recruits the other virion glycoproteins. HveA, when transfected in cells which do not express any other suitable receptor, mediates HSV-1 entry, but has such a narrow distribution that its actual usage is limited to very specialized cell types, e.g. T-lymphocytes.
The general aim of the present invention is to provide new agents of interest in the medical field for preventing, treating the HSV infections.
One of the objects of the present invention is to provide prophilactic and therapeutic agents to HSV-1, HSV-2 by the identification of a region of HIgR/PRR1 that is functional in HSV-1 entry and interacts with HSV-1 gD. Another object is to provide an anti-HSV agent, or antibody, specifically designated to block HSV-1 infections.
Yet another object is to provide cells lines resistant to infection by HSV-1, HSV-2 and BHV-1 for the biotechnological identification and production of proteins which act as mediators of HSV in human or animal models.
A further object of the present invention is to provide cells line expressing HIgR and other HSV-1, HSV-2 mediators for testing the efficacy of antiherpes drugs, antibodies and protein which interfere with virus entry.
In accordance with a first aspect of the present invention, a novel member of the immunoglobulin superfamily named HIgR that confers susceptibility to HSV infection by mediating entry of the virus into cells and to sequences encoding this immunoglobulin superfamily member is provided. The present invention is based, in part, by the unexpected evidence that the N-terminal domain, called V-domain, is the region of the molecule functional in HSV-1 and HSV-2 entry into cells and in binding to the virion glycoprotein D. Included within the present invention is the application of this new immunoglobulin portion (peptide) and sequences in the medical field.
Applicants have now found, in accordance with a first aspect of the invention, a novel member of the Ig superfamily, hereinafter named HIgR, which acts as receptor for HSV-1 and HSV-2 entry into human cells.
The present invention further provides a monoclonal antibody which is able to bind HIgR and is able to block infection with HSV-1, HSV-2 and BHV into human cells or into cells expressing HIgR as a transgene for the manufacture of a medicament to prevent or treat HSV-1, HSV-2 and BHV infections.
In accordance with a preferred embodiment of the invention it is provided the use of the V domain or its derivatives for the manufacture of a medicament for preventing or treating HSV-1, HSV-2 and BHV infections.
In accordance with an embodiment of the invention it is also provided the specific use of monoclonal antibody R 1.302 to HIgR for the manufacture of a medicament for preventing or treating infections of human tissues by HSV-1, HSV-2 and cell-to-cell spread of said virus to neighboring cells such as hepidermic or nervous cells. The derivation of humanized antibodies for use in the medical field is also provided.
In particular, Applicants have found the new member of Ig superfamily named HIgR by the provision of herein below disclosed J1.1-2 cell line which is highly resistant to entry of HSV-1, -2, BHV-1 and which was selected by repeated exposures of BHKtk-cells to a recombinant HSV-1 expressing tumor necrosis factor 1 (TNF-xcex1.). In accordance an embodiment of the invention, the screening of a human cDNA expression library for ones that restored susceptibility to J1.1-2 cells led to the isolation of a human cDNA one which encodes a novel transmembrane protein with features typical of the Ig superfamily and an overall molecular organization essentially overlapping that of the poliovirus receptor (PVR) (see Mendelsohn, C. L., E. Wimmer, and V. R. Racaniello. 1989. Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell. 56:855-65), i.e. a V-like and two C-like domains bracketed by conserved cysteins. Applicants have now also found that the major region of HIgR/PRR1 with HSV-1 entry activity resides in the V domain. In particular, the V domain is a major determinant of HIgR/PRR1 in mediating HSV-1 entry and is sufficient to mediate HSV-1 entry into cells. Furthermore, the single V domain is sufficient for the in vitro physical interaction with gD in a specific manner. The analyses of the interaction of gD with the functional regions of its receptor lead to practical and industrial applications of the present invention.
Since it is instrumental to define the interaction between gD and HIgR/PRR1 in terms of minimal size of the functional domain, structural requirements, key residues, etc, in the present application, the major functional region of HIgR/PRR1 involved in and sufficient for HSV-1 entry and able to physically interact with the viral gD has been selected and acknowledged as being a region of about 114 as residues.
A novel class of anti-HSV agents/medicaments designed specifically to block HSV-1 infection in accordance with an aspect of the present invention is provided by the identification that a major functional domain of HIgR/PRR1 is encoded in the V domain, coupled with the demonstration that this domain is sufficient for physical binding to gD and to compete with virion infectivity. A further embodiment of the present invention provides for the construction of transgenic mice expressing HIgR, or HIgR and PRR-2, or HIgR and other mediators of HSV-1, HSV-2 and BHV-1 entry from transgenes, for the purposes of establishing a mouse model for efficacy studies of antiherpes drugs, antiherpes antibodies, antiherpes vaccines, antiherpes proteins which interfere with virus entry.
A further embodiment of the present invention provides for the construction of transgenic mice expressing HIgR, or HIgR and PRR-2, or HIgR and other mediators of HSV and BHV-1 entry from transgenes, for the purpose of establishing a mouse model system for efficacy studies of genetically engineered herpes simplex viruses to be employed as vectors against tumors of CNS.
A further embodiment of the present invention provides for the construction of transgenic mice expressing HIgR, or HIgR and PRR-2, or HIgR and other mediators of HSV and BHV-1 entry from transgenes, for the purpose of establishing a mouse model system for efficacy studies of genetically engineered herpes simplex viruses debilitated in virulence genes, to be employed as vaccines.
A further embodiment of the present invention provides for the construction of transgenic mice expressing HIgR, or HIgR and PRR-2, or HIgR and other mediators of HSV and BHV-1 entry from transgenes, for the purpose of establishing a mouse model system for efficacy studies of genetically engineered herpes simplex viruses for efficacy studies of substances that interfere with establishment of HSV latency and reactivation of HSV from latency.
A further embodiment of the present invention provides for the construction of transgenic mice expressing HIgR, or HIgR and PRR-2, or HIgR and other mediators of HSV and BHV-1 entry from transgenes, for the purpose of establishing a mouse model system that sustains BHV-1 infection, the mouse model system allowing to study the efficacy of drugs, antibodies and vaccines. In accordance to a further aspect of the present invention is provided a medicament which blocks HSV-1 and -2 infection in humans, and limits the virus-induced lesions. HSV-1 and -2 can infect as free virions or by cell-to-cell spread of virus. This lattern pathway of transmission of virus is particularly relevant in humans, as in humans HSV replicates in tissues of muco-epithelial origin, like oral and genital mucosae and corneal epithelia, and then spreads to nerve endings of sensory neurons. Upon reactivation from latency, the virus replicates in sensory neurons, and moves in retrograde direction to muco-cutaneous tissues. Therefore, HSV spread through human tissues is mainly by cell-to-cell transmission, a mechanism that greatly contributes to shielding the virus from the humoral immune response.
In particular, Applicants has found that HIgR, and/or its splice variant isoform HveC, are involved in cell to cell spread of HSV from cell to cell in cell cultures, and in human cell lines.